• Pediatric Gastroenterology, Hepatology & Nutrition
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• v.4 no.1
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• pp.34-40
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• 2001

Purpose: The purpose of this study was to evaluate the clinical efficacy of reverse transcription-polymerase chain reaction (RT-PCR) in detecting rotaviral gastroenteritis in children comparing with that of commercial immunoassays. Methods: Stools from 79 children admitted Korea University Hospital due to diarrhea were collected from December 1999 to February 2000. Immunoassays were done using commercial rotavirus Latex kit and Rotatec (ELISA) kit. RT-PCR was performed to amplify group A rotavirus, most commonly pathogenic to human, using VP4- and VP7-specific primers. The detection rates of immunoassays and RT-PCR were compared. Results: ELISA assay was superior to LA assay and moderately concordant with RT-PCR in detecting rotaviral gastroenteritis. Conclusion: Although RT-PCR is known very sensitive, it does not have significant advantage over immunoassay in detecting rotaviral gastroenteritis.

• Proceedings of the Microbiological Society of Korea Conference
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• 2008.05a
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• pp.171-171
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• 2008

We monitored the occurrence of human enteric viruses in urban rivers by cell culture-PCR and RT-nested PCR. Water samples were collected monthly or semimonthly between May 2002 and March 2003 in four urban tributaries. Enteric viruses were detected by RT-nested PCR and cell culture-PCR based on a combination of Buffalo Green monkey kidney (BGMK) and A549 cell lines, followed by phylogenetic analysis of amplicons. By RT-nested PCR analysis, 45 (77.6%), 32 (55.2%), 32 (55.2%), 26 (44.8%), 12 (20.7%), 2 (3.4%), 4 (6.9%), and 4 (6.9%) of 58 samples showed positive results with adenoviruses, enteroviruses, noroviruses (NV) genogroup I (GI) and II (GII), reoviruses, hepatitis A viruses, rotaviruses and sapoviruses, respectively. Adenoviruses were most often detected and only eight (13.8%) samples were negative for adenoviruses and positive for other enteric viruses in the studied sites. Thirty-one (77.5%) of the 40 samples were positive for infectious adenoviruses and/or enteroviruses based on cell culture-PCR, and the frequency of positive samples grown on A549 and BGMK (65.0%) was higher than that grown on BGMK alone (47.5%). The occurrence of each enteric virus, except reoviruses and hepatitis A viruses was not statistically correlated with the water temperature and levels of fecal coliforms according to Binary logistic regression model. By sequence analysis, most strains of adenoviruses and enteroviruses detected in this study are similar to the causative agent of viral diseases in Korea and most NV GI- and GII-grouped strains were closely related to the reference strains from China and Japan, and GII/4-related strains had similar sequences to strains recognized as a worldwide epidemic outbreak. Our results suggested that monitoring human enteric viruses is necessary to improve microbial quality and cell culture-PCR using the combination of A549 and BGMK cells and the adenovirus detection by PCR could be useful for monitoring viral contamination in the aquatic environment.

• The Korean Journal of Mycology
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• v.20 no.4
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• pp.315-323
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• 1992

The effects of several parameters on PCR amplification in using RAPD were studied. The results of this study suggest that approximately 15 ng of genomic DNA in $20\;{\mu}l$ of reaction mixture results in discrete and reproducible PCR products. In addition, the results indicate that concentration or amounts of reaction components studied are highly inter-dependent in their effects, and RNA can interfere severely with PCR amplification. Suitable concentrations or amounts of reaction components were found to be 30 ng of 10-mer primer, $200\;{\mu}M$ of dNTP, 0.001% gelatin 1.5 mM $MgCl_2$, 10 mM Tris-Cl (pH 8.8), 50 mM KCl, 0.1% Triton X-100, 2 units of Taq DNA polymerase, and 15 ng of RNase-treated genomic DNA in $25\;{\mu}l$ of reaction mixture.

• Korean Journal of Veterinary Research
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• v.38 no.1
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• pp.173-181
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• 1998

Esherichia coli O157 : H7의 slt I, slt II, uid A 및 eaeA 4종 유전자를 동시에 검출하기 위한 multiplex PCR 기법을 확립하고 쇠고기중 직접 E coli O157 : H7 검출시험을 실시하였다. 4 set의 primers를 이용한 multiplex PCR 기법으로 31종의 장내세균에 대한 특이성을 조사한 결과 E coli O157 : H7 에서 1,087bp (eae A), 584bp (slt II), 348bp (slt I) 또는 252bp (uid A)크기의 DNA를 동시에 특이적으로 검출할 수 있었다. E coli O157 : H7 15주는 모두 uid A 및 eae A 유전자가 동시에 검출되었고, 다른 장내세균에서는 검출되지 않았다. slt I 또는 slt II 유전자를 가지고 있는 E coli 표준균주 24종을 이용하여 multiplex PCR 기법과 Vero cell cytotoxicity assay을 비교검사한 결과 베로톡신 산생능과 PCR법의 결과는 일치하였다. mutiplex PCR 기법의 쇠고기중 검출한계는 modified EC(mEC)에서 증균없이는 E coli O157 : H7균 $10^4cells/g$ 이상에서 검출이 가능하였으나 mEC에 1차 증균후 modified TSB 증균하였을 경우에는 10cells/g이하까지도 검출이 가능하였다. 개발된 multiplex PCR 기법을 쇠고기 40종에 직접 적용한 결과 E coli O157 : H7은 검출되지 않았으나 slt I 및 slt II유전자를 가지고 있는 E coli 4종이 검출되었으며, 이들의 혈청형은 O6, O112, O115 및 O139 였다. 이 연구에서 개발된 multiplex PCR은 쇠고기중 E coli O157 : H7을 신속하고 특이적으로 검출하는데 사용할 수 있을 것으로 사료된다.

• Korean Journal of Food Science and Technology
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• v.36 no.5
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• pp.723-727
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• 2004

Qualitative and quantitative PCR methods were performed to examine detection and quantitation of epsps inserted into genetically modified soybean (GMS) in processed foods, soy milk, tofu, and biji (soybean curd residue). Using PCR amplification to produce two (121 and 330 bp) epsps in GMS, detection limits of GMS in soy milk, tofu, and biji containing 0.01% GMS were measured. For quantitative detection, test samples containing 1, 3, and 5% GMS were measured by real-time PCR method. Results show real-time PCR method is applicable to detect GMS quantitatively in processed foods.

• Journal of Korean Society of Forest Science
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• v.95 no.6
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• pp.631-635
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• 2006

Single-strand conformation polymorphism (SSCP) analysis of MD and JWB phytopalsma isolates which amplified PCR products using the R16F2n/R2 phytoplamsa universal primer pair were compared for variations of their nucleotide sequence. The MD and JWB phytoplasmas were clearly distinct each of the band patterns from about 1.2 kb PCR products. To clearly distinct of close SSCP band patterns, the MD and JWB phytoplasma PCR products were mixed and performed to detect their polymorphism. The SSCP band patterns show all of bands of MD and JWB on single lane and easily distinct their each band patterns. The PCR-SSCP analysis was possible to detect of 1.2 kb nucleotide sequence and near close band patterns were easily distinct by mixing two samples.

• Journal of Plant Biotechnology
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• v.5 no.2
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• pp.83-86
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• 2003

A reverse transcription polymerase chain reaction (RT-PCR) protocol was developed using two specific 22-mer primers located in coat protein gene of SPFMV. A 411 bp PCR-product was detected in virus infected plants as well as tissue culture raised sweet potato but not in healthy plants. For optimization of RT-PCR protocol, the optimum crude nucleic acid concentration, annealing temperature, primer concentration and numbers of PCR-cycle for maximum sensitivity and specificity were determined. The optimum condition for RT-PCR was as follows: RT-PCR reaction mixture was one-step mixture, containing 50 pmol of primer, 30 units of reverse transcriptase, 5 units of RNasin, and the crude nucleic acid extracts (200 ng). In RT-PCR, cDNA was synthesized at $42^{\circ}C$ for 45 min before a quick incubation on ice after pre-denaturation at $95^{\circ}C$ for 5 min. The PCR reaction was carried out for 40 cycles at $96^{\circ}C$ for 30 see, $63^{\circ}C$ for 30 sec, $72^{\circ}C$ for 1 min, and finally at $72^{\circ}C$ for 10 min. The viral origin of the amplified product was confirmed by sequencing, with the sequence obtained having $95-98\%$ homology with published sequence data for SPFMV. The benefits of this RT-PCR based detection of SPFMV would be simple, rapid and specific.

• Parasites, Hosts and Diseases
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• v.53 no.2
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• pp.147-154
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• 2015

Various constituents in clinical specimens, particularly feces, can inhibit the PCR assay and lead to false-negative results. To ensure that negative results of a diagnostic PCR assay are true, it should be properly monitored by an inhibition control. In this study, a cloning vector harboring a modified target DNA sequence (${\approx}375bp$) was constructed to be used as a competitive internal amplification control (IAC) for a conventional PCR assay that detects ${\approx}550bp$ of the Cryptosporidium oocyst wall protein (COWP) gene sequence in human feces. Modification of the native PCR target was carried out using a new approach comprising inverse PCR and restriction digestion techniques. IAC was included in the assay, with the estimated optimum concentration of 1 fg per reaction, as duplex PCR. When applied on fecal samples spiked with variable oocysts counts, ${\approx}2$ oocysts were theoretically enough for detection. When applied on 25 Cryptosporidium-positive fecal samples of various infection intensities, both targets were clearly detected with minimal competition noticed in 2-3 samples. Importantly, both the analytical and the diagnostic sensitivities of the PCR assay were not altered with integration of IAC into the reactions. When tried on 180 randomly collected fecal samples, 159 were Cryptosporidium-negatives. Although the native target DNA was absent, the IAC amplicon was obviously detected on gel of all the Cryptosporidium-negative samples. These results imply that running of the diagnostic PCR, inspired with the previously developed DNA extraction protocol and the constructed IAC, represents a useful tool for Cryptosporidium detection in human feces.

• Korean Journal of Environmental Biology
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• v.35 no.4
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• pp.468-475
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• 2017

This study is for the consideration of the existence tendency of Kudoa septempunctata in olive flounder. In general, muscle has shown a strong PCR positive reaction in spores containing tissues rather than non-containing tissues. However, blood PCR results showed opposed tendency. In various organs of the tested fish containing spores in muscle tissue, heart had shown positive reaction along with muscle at PCR analysis. Muscle fiber necrosis was observed at the histological observation, and this degeneration was common in both samples. The one sample was the PCR positive muscle containing spore and the other was the PCR positive muscle non-containing spore. Both of muscle tissues indicated a positive reaction at ISH (in-situ hybridization) against K. septempunctata.

• The Journal of Korean Obstetrics and Gynecology
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• v.27 no.1
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• pp.17-27
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• 2014

Objectives: This study was conducted to evaluate the inhibitory effect of polygoni cuspidati radix (PCR) extract on osteoclast differentiation. Methods: MTT-assay was performed to estimate cytotoxicity of PCR extract in BMMs stimulated with RANKL. Tartrate resistant acid phosphatase (TRAP) staining, TRAP activity and RT-PCR were performed to know the inhibitory effect on osteoclast differentiation. actin ring formation were analysed to observe the effect of PCR extract. Results: PCR decreased the number of TRAP positive cells and TRAP activities in BMMs stimulated with RANKL and M-CSF. PCR restrained the formation of actin ring. PCR down regulated the induction of NFATc1, c-Fos, TRAP and OSCAR by RANKL. PCR inhibited NF-${\kappa}B$ activity by inducing degradation of $I{\kappa}B{\alpha}$. Conclusions: We suggest that PCR Extracts can be an effective therapeutic agent on osteoclast differentiation caused by diseases such as osteoporosis.